(A) Experimental Laboratory Component: Using reverse genetics, low pathogenic avian influenza (LPAI) viruses (H5N1 and H6N1) with the minimal multibasic cleavage site (MBCS) motif will be generated and the intravenous pathogenicity index (IVPI) and 50% bird infectious dose (BID50) will be determined in chicken and compared to LPAI H5N1 and H6N1. Next, these viruses will be serially passaged (10x) in chicken to see whether the minimal MBCS will evolve into a MBCS and the highly pathogenic avian influenza (HPAI) phenotype. Deep sequencing (RML Research Technologies Section, Genomics Unit) will be used to analyze the cleavage site motif of viruses from chicken in each passage. Note - This part of the project is still on hold due to the effects of the H5 moratorium and subsequent restrictions by NIAID, NIH. No work has been done on this project in fiscal year 2013/14. Instead we have evaluated in collaboration with Dr. Subbarao's group the replication of wildtype (wt) and cold-adapted (ca) viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of African green monkeys (AGMs). All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates. (B) Field Survey Component: Historically, the natural reservoir for all influenza A viruses were considered wild birds. However, the recent identification of a novel H17 influenza A virus in yellow-shouldered bats in Guatemala has put the focus on novel reservoirs besides wild birds. Certain influenza A viruses in particular are a direct threat to both animal and public health. In particular, Highly Pathogenic Avian influenza A viruses (HPAI H5 and H7 subtypes) and a wide variety of swine influenza viruses (H1, H2 and H3) are notorious for crossing the species barrier from animals to humans. Public health, animal health, agricultural productivity and food security in the Republic of Congo (RC) and the wider Congo basin region are directly threatened by outbreaks of influenza A viruses. In addition, the potential zoonotic transmission of influenza viruses to the human population could spark outbreaks and the emergence of a new pandemic. To date little or no information has been published on the prevalence, incidence and identity of animal influenza viruses, in particular avian influenza viruses, in RC and there are currently no surveillance programs for avian influenza viruses in place. Therefore, there is a real need to develop baseline information with reference to the circulation of avian influenza viruses. During the fiscal year 2012/13, the laboratory infrastructure has been established at the Laboratoire National de Sant Publique in Brazzaville (RC capital) and diagnostic/surveillance assays have been developed and implemented. Surveillance studies have continued over the 2013/14 funding period and results are currently analyzed and prepared for publications. This project will be continued by Dr. Vincent Munster, Viral Ecology Unit, LV, NIAID, NIH. Dr. Feldmann will no longer serve as the principal investigator for this project.